Capillary electrophoresis is a separation method employed in analytical chemistry which utilizes the differences in electrophoretic mobility of the sample substances to be separated. Capillary electrophoresis is used, for example, for separating different biological molecules, such as proteins or peptides. The separation process is performed in a capillary tube which is typically open on both ends and to which an electric field is typically applied which causes electrophoretic separation of different sample substances within the capillary tube. The electric field is applied by means of electrodes which are arranged at the ends of the capillary, respectively, and which are connected to a high voltage power supply. The capillary is filled with an electrically conductive electrolyte so that an electric field can build up within the capillary. The two ends of the capillary are immersed in vials containing the electrolyte, respectively.
When new sample substances are to be introduced into the capillary for subsequent separation, the vial containing the electrolyte is removed from one end of the capillary, a vial containing the sample is positioned at this place so that the end of the capillary is immersed in the sample liquid. Thereafter, the sample is injected into the capillary. In the state of the art, basically two different methods for sample injection have been developed: the first method is based on electromigration, and the second method is based on pressure differences between the first and the second end of the capillary tube.
According to the injection method based on electromigration, an end of the capillary and an electrode are placed into the sample and a voltage is briefly applied which causes an amount of sample liquid to electromigrate into the capillary under the action of the applied electric field. A disadvantage of this method is that the injection already causes a preseparation of the sample components due to different ionic strength and resulting electrophoretic mobility of the sample substances. A further disadvantage is that electrochemical by-products may be created which may contaminate sensitive samples.
According to the injection method based on pressure differences, the sample is forced into the capillary by an outer pressure difference which may be created either by positioning the outlet end of the capillary at a different level than the inlet end, or by applying overpressure at the inlet end or underpressure at the outlet end of the capillary. A method of the first type wherein inlet and outlet ends of the capillary can be positioned at different levels for the injection step is known from the article D. J. Rose and J. W. Jorgenson, "Characterization and Automation of Sample Introduction Methods for Capillary Zone Electrophoresis" Analytical Chemistry 60 (1988), pages 642-648. This approach has some disadvantages. First, an apparatus using this injection method requires a comparatively large space since the capillary has to be moved over a certain distance. Second, the protection of the user against high voltage and the removal of heat from the capillary generated during electrophoresis require considerable expenditure due to the mobility of the capillary.
According to a further known injection method, sub-atmospheric pressure is used to introduce the sample. For this purpose, the outlet end of the capillary where the detector is located comprises corresponding arrangements for generating sub-atmospheric pressure. This has the disadvantage that it is difficult or impossible to couple further analytical instruments and/or detectors to the outlet of the electrophoresis apparatus. Furthermore, the known method uses a comparatively large underpressure for the injection which leads to very small injection times. As a consequence of these short injection times, the accuracy of the electrophoretic separation is impaired. In order to explain this, some general remarks on sample injection will now be made.
The purpose of an injection is to introduce a quantitatively defined volume of sample into the separation capillary. The relationship between the flow in a tube and the pressure difference is described by the law of Hagen-Poisseuille: In a capillary tube of a given length and internal diameter, the flow is proportional to the pressure difference and inversely proportional to the viscosity of the liquid. For a sufficiently short time, for example a couple of seconds, the viscosity can be considered constant so that the flow is proportional to the pressure acting on one end of the capillary if the pressure at the other end is constant, for example ambient pressure. From the above it follows that in case of a constant pressure the injected volume is proportional to the time during which the pressure acts. In case of a variable pressure, the injected volume is proportional to the time integral of the pressure curve. For a given pressure, the injection time becomes smaller if the sample volume to be introduced becomes smaller. For very small injection times, the switching and delay times of the hydraulic and pneumatic components become relevant so that the quantitative accuracy of the electrophoretic separation is impaired. This explains the disadvantages of the above mentioned prior art method. A further limitation of the pressure injection methods arises at low pressures if the velocity of the liquid which is caused by the application of the pressure, is in the range of the diffusion velocities of the sample components. If this is the case, the selectivity of the electrophoretic separation is impaired.
A further prior art injection device is disclosed in EP-A-0 475 533. This known device does not use a permanent pressure source but a kind of piston pump which is brought in relationship with the vial containing the sample liquid. When pushing the piston inwardly, the gas pressure in the sample vial increases so that sample is forced into the capillary. In order to achieve a high injection accuracy with this device, it is important that all components are well sealed against the environment and that all seals are durable and reliable. Otherwise, the injected volumes would be dependent on the ambient pressure so that fluctuations of the ambient pressure would lead to measuring errors.
From EP-A-0 339 781, an injector for a capillary electrophoresis apparatus is known wherein overpressure is applied to the liquid to be injected at the end of the capillary distant from the detector. The pressure applied is substantially constant during the injection. At the beginning of the injection, the pressure value quickly jumps to the desired constant value. The pressure is measured by a pressure sensor and integrated with respect to time to derive the flow rate for determining the volume introduced into the capillary. By comparison of the measured injection volume with the desired volume entered by a user, control signals for controlling the valves of the injector are produced. Such a control signal causes the switching off of valves at the end of an injection period. In response thereto, the pressure drops again to ambient pressure, but this decrease occurs passively, without active pressure control so that it may come to fluctuations from one injection to another. Such fluctuations may be caused, for example, by changes in the ambient pressure or by temperature changes. Furthermore, the specifications for the components of the injector might be different for different devices which also leads to unwanted deviations. For small injected volumes, the accuracy may be impaired. Furthermore, the abrupt application of the pressure at the beginning of an injection may cause sloshing around and splashing of the sample liquid which may lead to sample loss and/or to a higher evaporation rate of the carrier liquid and thus to a change in the sample concentration.